An important stage in doubled haploid (DH) production is to evaluate and to differentiate the ploidy level of regenerant plants at least two–three times during the technology. Therefore, rapid and reliable methods are necessary for particular species taken into the technology. In this study, Cucurbita pepo regenerants obtained through unpollinated ovule culture in vitro were evaluated including three different methods: direct chromosome counting in apical meristems, flow cytometry of the cell nucleus, and estimation of morphological parameters of the abaxial epidermis. Methods were optimized for each of three evaluations, and main criteria were determined for ploidy level differentiation. As a result, four ploidy levels, namely, 2n, 3n, 4n, and 8n, were defined among regenerant plants adapted to ex vitro conditions, while true haploids were only found among plants that remained in the in vitro culture. In total, 32.35%, 26.47%, 33.82%, 4.41%, and 2.94% of regenerant plants of courgette and patisson were diploid, triploid, tetraploid, octaploid, and aneuploid, respectively. According to results of flow cytometry of the cell nucleus, two cytotypes in diploid samples with DNA content of 2C = 1.07 ± 0.03 pg for courgette belonging to subsp. pepo and 2C = 0.95 ± 0.03 pg for patisson samples belonging to subsp. ovifera were revealed. The images of metaphase chromosomes of haploid, triploid, and tetraploid C. pepo specimens obtained using the propion–lacmoid chromosome staining method were presented for the first time. Parameters of abaxial epidermis in diploid samples of courgette and patisson grown in open-field and greenhouse conditions were described and compared. It was shown that the most robust parameter not depending on external factors was the number of chloroplasts in stomatal guard cells, which contained 9.41 to 11.31, 14.84 to 16.3, and up to 17.58 chloroplasts in diploid, triploid, and tetraploid samples, respectively. The application of several methods for estimation enables avoiding the misidentification of ploidy levels in adapted regenerant plants produced with the use of DH technology.

Doubled haploids have been widely used worldwide in breeding programs and fundamental research as valuable homozygous material for about 100 years. The species Cucurbita pepo L. are represented by a huge variety of forms, include highly productive vegetable crops and have a wide distribution in the world. Despite the great economic importance, the creation of effective protocols to ensure stable production of doubled haploids in this species remains an urgent task. DH plants are of interest not only because of the acceleration of the breeding process, but also because of the realization of the huge potential of gametoclonal variability inherent in this highly polymorphic species. In this review, we analyzed the main technologies used for obtaining doubled haploids in vegetable crops of C. pepo: parthenogenesis in situ stimulated by treated/irradiated pollen, gynogenesis in vitro (unpollinated ovule culture in vitro) and androgenesis in vitro (anther/microspore culture in vitro). An analysis is presented of the research carried out from the beginning of the discovery of haploid plants to the current advances and evaluation of the prospects in the field of DH plant production. The main critical factors influencing the efficiency of each technology and its individual steps are considered. The developed technology of doubled haploids obtaining using non-pollinated ovary culture in vitro is presented. This technology allows to obtain up to 55 embryoids per one cultivated ovary (28 embryoids/ 100 cultivated ovules) To introduce haploid technologies into the breeding process it is necessary to evaluate the obtained plants for ploidy level. The use of direct counting of chromosomes in apical cells may present a certain difficulty in this species due to their large number (2n=40) and their small size. Depending on the level of laboratory equipment, ploidy determination using flow cytometry of cell nuclei and counting the number of chloroplasts in stomatal guard cells in the epidermis of the abaxial side of the leaf may be more convenient methods. The prospects for the use of molecular markers for assessment for homozygosity in DH technologies used, including C. pepo, are discussed in the review.

Relevance. To create an effective technology for obtaining doubled haploids (DH-technology) of zucchini in unpollinatedseedpod culture in vitro it is necessary to select the optimal values of many factors, the degree of influence of each of which on gynogenesis can vary significantly. The aim of the study was to optimize the individual stages of the technology.Methods. Liquid and agarized (7 g/L) IMC medium with different sucrose concentrations (20 to 80 g/L) and different plant growth regulators (2 mg/L 2,4 D; 0.2 mg/L TDZ ; 0.8 mg/L 2,4 D and 1.2 mg/L NUC) were used for induction of embryogenesis.Results. Optimal for the studied zucchini genotypes was pre-isolated from the evening, plucked in the morning opened bud. Sterilization of zucchini ovaries by short-term burning after treatment with 96% alcohol, allows significant reduction of the time required for this step without loss of embryogenic potential. IMC nutrient medium with sucrose (20 to 40 g/l) can be used for induction of gynogenesis in the unpollinatedzucchini ovary culture. The use of nutrient media with 2 mg/l 2,4 D for most genotypes was more effective and resulted in higher number of embryoids compared to nutrient media containing 0.2 mg/l TDC and media with 0.8 mg/l 2,4 D and 1.2 mg/l NAA. Embryoid formation was observed after 5 weeks of cultivation.Conclusion. We were able to complete the full cycle of technology for obtaining doubled haploids in unpollinatedseedpod culture in vitro for 30 zucchini genotypes and obtain DHplants, which are valuable source material for both breeders and genetic research. Optimization of the individual steps of the technology made it possible to achieve the maximum result for individual genotypes – 55 embryoids per 100 cultivated ovules.

Relevance. In accordance with the needs of the market, in 2008, a variety of zucchini Russian spaghetti with two-colored fruits was created. We faced difficulties in maintaining a high percentage of two-color forms in the variety population. To solve this problem, we studied the possibility of using markers of young zucchini leaves in the selection of forms with different fruit colors in technical ripeness.Material and conditions. The experience was started in 2005 to 2018 in the open ground on the basis of FSBSI FSVC. In the breeding nursery, research was conducted annually on 30 plants. The color of the fruit was taken into account only in the phase of technical ripeness. The best plants were propagated by incest. In the nursery breeding varieties of Russian spaghetti were sown by family. During 4 years in each family, at different stages of development, 50-100 plants were studied according to the color of the fruit and other economically useful characteristics.Results. For eleven years, incuchination and selection were carried out on two-color forms of zucchini Russian spaghetti to achieve homozygosity and, accordingly, to align the material with the color of the fruit. Some regularities of the influence of the number of integrirovanii on the color of the fruit has not been observed. Continuing to observe the plants, it was possible to establish a relationship between the color of the fruit in technical ripeness and the pattern on the lower (first) leaves. On plants with a marble pattern, the leaves were mostly green (reticulated); with yellow spots on the leaves – two-colored and with completely yellow leaves – yellow fruits. By selecting plants with yellow spots on the leaves in the early stages, in the phase of the 3-4-th real leaves, we were able to increase the percentage of plants with two-colored fruits in the population of the variety to 95.5-100%. All this greatly simplified the original seed production of zucchini Russian spaghetti.

Relevance.It is very important not only to create a hybrid of cucumber, which in all respects suits the producers of vegetable products, but also to ensure that the production of hybrid seeds with high varietal and sowing qualities is not unprofitable. A high yield of hybrid seeds can be obtained only with good pollination of the maternal line, for this purpose there should be enough male flowers on the paternal form and the flowering time of the parent forms should coincide.Materials and methods. The research was carried out in the first turn of the winter greenhouse with low-volume cultivation technology in 2017-2018. The object of research was the parent forms of a promising hybrid of parthenocarpic type F1 Murava. The maternal form is of the female type, the paternal form is predominantly of the female type of flowering. Terms of sowing of parental forms were studied: simultaneously and with a difference of 7-8 days. Experiments on different variants of treatments of the paternal form with gibberellic acid (concentration 0.09-0.1%) were laid in order to obtain a sufficient number of male flowers for pollination of the maternal form. The parent forms were planted at the hybridization site according to the 3:1 scheme. Pollination of female plants was carried out manually daily.Results. As a result, it was concluded that for good pollination of the maternal form of the hybrid F1 Murava, two triple treatments of the paternal form with gibberellic acid in the phase of two real leaves are needed. Treatment with gibberellic acid paternal form, sown a week later, was more effective. Obviously, this is due to the improvement of growing conditions at a later date of planting seedlings. When sowing the mother form a week later than the father, the seed productivity of hybrid seeds was higher, compared with simultaneous sowing of parent forms and reached 53.5 g per plant.